Chitin and chitin derivatives for promoting wound healing

ABSTRACT

Wound healing compositions and the process of healing wounds with such compositions are described, the compositions containing chitin, partially depolymerized chitin or a chitin derivative.

United States Patent 1191 Balassa [111 3,903,268 1 51 *Sept. 2, 1975 CHITIN AND CIIITIN DERIVATIVES FOR PROMOTING WOUND HEALING [75] Inventor: Leslie L. Balassa, Blooming Grove,

[73] Assignee: Lescarden Ltd., Goshen, NY.

[ Notice:' The portion of the tenn of this patent subsequent to Jan. 4, 1989, has been disclaimed.

22 Filed: Feb. 19, 1971 21 Appl. No.1 117,085

Related US. Application Data [60] Division of Ser. No. 704,538, Feb. I2, 1968, Pat. No.

3,632,754, and a continuation-in-part of Ser. No. 619,007, Feb. 27 1967, abandoned.-

52 us. Cl. 424/180; 424/28; 424/95; 424/154 51 Int.Cl. A6lK27/00 [58] Field of Search 424/28, 95, 180, 154

Carlozzi et al.... 424/180 Weisberg et a]. 424/330 Primary Examiner-Stanley J. Friedman Assistant ExaminerDaren M. Stephens Attorney, Agent, or Firm-Darby & Darby [57] ABSTRACT Wound healing compositions and the process of healing wounds with such compositions are described, the compositions containing chitin, partially depolymerized chitin or a chitin derivative.

9 Claims, No Drawings scribes l CHITIN AND CHITIN DERIVATIVES FOR PROMOTING WOUND HEALING This application is a division of my copending application Ser. No. 704,538 filed Feb. 12, 1968 now US. Pat. No. 3,632,754 and a continuation-in-part of copending application. Ser. No. 6l9,007 filed Feb. 27, 1967 now abandoned.

This invention relates to methods of promoting the healing of wounds and compositions therefor comprising chitin, and/or chitin derivatives and/or partially depolymerized chitin.

Medicine has long been interested in improving the H healing of wounds. Patients suffering from diabetes or undergoing extensive cortisone treatment show extremely slow rates of healing of any wounds which they receive. Thus, surgery on such patients involves additional risks not present with other patients. Moreover,

rapid healing of wounds is particularly desired for patients in tropical countries'where the risk of infection is high. Rapid healing is also desired in the case of soldiers who have been wounded in a battle zone and cannot easily and quickly be removed therefrom. Acceleration of wound healing is highly desirable in the case of patients who cannot readily be immobilized, such as farm animals.

In evaluating the utility of a material to promote wound healing, a reproducible test is necessary to give treated rat and the control rat is expressed as the percentage improvement obtained. Considering biological variance it is believed that only differences of about 10% or more are significant.

There have been several recent developments reported concerning materials which promote wound healing. in this connection US. Pat. No. 3,232,836 dethe parenteral administration of N- acetylglucosamine as a wound healing material. Utilizing the test methodof Prudden et a] referred to in the preceding paragraph, N-acetylglucosamine showed improvement in tensile strength of only about 10% whereas Prudden and his co-workers have reported significantly larger increases in wound healing by the use of cartilage preparations from various animals. Depending on the age and species of animal and the fineness of the cartilage powder, improvements ranging from to 40% in wound healing tensile strength have been reported by Prudden.

Now it has been discovered that finely divided chitin, partially depolymerized chitin, and chitin derivatives possess the ability to promote the healing of wounds.

Accordingly, one aspect of the present invention relates to novel methodsof promoting and assisting the ;healing of wounds as, for example, damaged mammalian tissue, open ulcers, etc.,and to compositions therefor.

Another aspect of the invention relates to significant .improvements in wound healing strength achieved by the administration of finely divided chitin, partially depolymerized chitin or chitin derivatives to a patient.

An additional aspect of the present invention is concerned with articles of manufacture such as surgical bandages, surgical sutures, etc., containing the wound healing materials of the present invention.

These and other aspects of the present invention will be apparent from the following description.

Chitin is a polysaccharide, believed to be poly (N- acetylglucosamine) which forms the cell walls of fungi and the hard shell of insects and crustaceans. As used herein, the term chitin embraces naturally occurring chitin synthetic chitin, as well as poly (N- acetylglucosamine) and its epimer poly (N- acetylgalactosamine). The N-acetylated partially depolymerized chitin, e.g. chitotriose, chitobiose, is a substance which retains its polymeric nature but has undergone a reduction in molecular weight (i .e. chain length) as a result of (l) enzymatic action such as by a chitinase enzyme, (2) chemical treatment such as acid hydrolysis or alkaline treatment, and (3) physical treatment. These materials are known in the art and procedures for their preparation may be found in Advances in Carbohydrate Chemistry" Vol. 15, Pages 380 to 384, Academic Press, New York 1960, the disclosure of which is incorporated herein by reference. Thus, the molecular length is in the range from 11-1 in which n corresponds to the number of repeating units in chitin to n=0 which is acetylated chitobiose.

The chitin derivatives contemplated are materials such as ethers formed with pharmaceuticailyacceptable radicals and esters or salts with pharmaceutical]y-acceptable' acids. Examples of suitable derivatives include hydroxy lower alkyl chitin such as hydroxyethyl chitin, carboxy alkyl chitin such as carboxymethyl chitin, salts of carboxy lower alkyl chitin such as the zinc salt, lower alkyl chitin such as methyl chitin and ethyl chitin, chitinacetate, chitin nitrate,

chitin citrate, chitin phosphate, N-acylderivatives derived from monocarboxylic aliphatic acids such as N- tained with the chitin materials is at least equal to and in many instances greater than that derived from the cartilage materials of theprior art. The substantial immovement in rate of healing which is obtained from the use of poly (N-acetylglucosamine), i.e., chitin, ascompared to-monomeric N-acetylglucosamine is particularly surprising. As compared to the great variability in cartilage depending on the animal, itsiage and the method of collecting the cartilage, chitin, particularly chitin of fungal origin, is a relatively uniform and easily obtained material.

The compositions of the present invention are applied using the same techniques and processes developed for cartilage, and N-acetyl'glucosamine. Thus, it is preferred to topically apply finely divided chitin directly to the wound surface. However, tablets, capsules or pellets of chitin may be prepared from mixtures of chitin, partially depolymerized chitin or chitin derivatives with well-known pharmaceutical excipients such as starch, sugar, certain forms of clay, etc. Such tablets, capsules or pellets may be taken orally or implanted nearrthesitus pfthe wound f lter natiyely, acolloidal soleut may be prepared-fromchitin,preferably iri isb eferablyin isotonic saline solution, and

A poyvder orsolutioh of chitiriorof a chitin deriv a- 1 tive mayalso be used to impregnate a surgical gauie. 0r pad whichis applied to the wo und. Chitin may also. be

a tissplv a hw k l -sh t n xantl teewun ptq fi er tonic saline, ra Wateresoluble, d erivative of chitin may ls tered,intramu scularly, parenterally 4 ns yfiivids PhiF I -QJ? it n,t e iz i eim b pplied topically by blo a meterqe'd amountpfithe material onto the using a hand at'o n riiz er Alternativelyfit may be applied by dusting as from a hand shaker or rna y beplac ed" together :With an inert gas under increasedpressure (i,e., above atmospheric pressure) in apressure vessel. In this latter means of appli and regeneratedasthe yirtually undeg raded polymer in' accordance: .y vjth tli'ei proc edures described in the prior .art by 'lihor et algPartially deace tylated chitin filaments vand fibers maybe. prepared in accordance with the procedgre described in, wisht, No. 2,040,880, These Asl v sa sly wd whe h il n $1 1 Pl injecltioniile either intramuscularly, parenterallyor ,-in,trav en9usly ,,it is first necessary to prepare adisperham 9% ,sg u en 9f h.?- ma i s; a ic l y acceptable, liquid Colloidal solutionsof chitin may be ptepa red sjng theernethod described by Lingappa and Loic lgyvqod l $9,,page 158 (1961). When adniinist redintravenously it is preferred to administer mthe cornpound in isotonie solution such as isotonic saegline, Y

Teh s i Hi0 ,jadrnixt re withgeachother, cartilage, or maybe ,co-adrninistered with other -therape u tically effective a e t wshfissss asid s r ylpelm a P rma ceut i cally aceept-able zi ncsalts-such as zinopggide,

m ne a s'z bm t nch hl te an z n e fisa at; ai isc tics such as thirnerosal and benzalkonium chloride; .zlolgal anesthetics sueh as lidogaine and procaine; antibiotics,suqlzy;as chloramphenicol, sulfanilairhide and amepi'eilline Qonjbinations ofthe therapeutically effective ,-,agents described above ohitinand/or, chitin deriya-' -ti,ve s may;b e,-,=u sed.Q I 4 f 1 ,Suitablesources of ,chitin are from lobsters, shrimp -;,.andhoth;e nustacea To. utilize. chitin frorn. such sol,tr oe ,;i necessary .to reduce the chitin in particle izetqlessghan about {1.59 microns and preferably less QthamabOut S O rnicr ons. Due to the tough andrather fibrousnature of chitin frorn such sources, this grinding {'.is di ffiqultand ex'pen'siye Accordingly, it is preferred to use;chitin'of fungalorigin. The cell walls of fungi are .g i adf t n- 1th stoeextraot ,the

v thylene oxide the, entire fungal mat pro- .s i m he .ndh iit 'ne m ri s thus substane een fonnd ,th t t -is not necessary v mn: frorn the rern l'IIIfIg CCIl material. Thus, ifdesir ed', after suitable ster'ilizationas byheat or cation, termed fae rosol application, the finely divided chitin pr chitin optionally, with other :medicamentsas indicated, may bepackaged as a dry aerosol powder ,as described in Dutch Pa't." application 6,415,252, published July 5, l95 this patent applijcation' is directed to a medicament for mastitis but the method of aerosol packaging described is applicable to powdered medicament haying describedp'article size) or as an aerosol foarn.

' 1h the following exampl thefwoundfhealing :efficiency of the yarious chitinous materials is 'deterrnined by, usingthe methdddf :Prudden et alas' de'sc'rib ed above. ln general','atlestflQ pairs of r'a'ts are used to obtain a meaningful ayera g'e for each material tested.

EXAMPLE 1 Co minercial lobster shell chitin jis grdundito a 'fine weight ratio of 1 chitin to 2 pebbles. Dry ice is then put on top of the m'ill charge and the mill is open for 5 minutes to allow the co, to; displace'thdair'in the in. The he f h rnill is then clamped 6 tight and'tl ie ig iinding'carr'idoutfdr'96 hours.Approiirnat'ely 50% r-chitindc-tiyatiyesn ay be used alone, in

;er' thepon/dered chitinipasseddthrough micron I he whole powdered chitin sci produ ced is 'the'n applied tothe 45, t es t' rats of 45 pairs of rats' 'used" in the Prudden et ai assay method described above, cent of wound healing for the treatediat's, stating the control rats as IQ( is 122%, i.e., theuse of chitin re- Various fungi are grown on either bfain-heart infusion (200 gm. calf brain, 250 beef heart, gin.

, prqteose pep tone:,'2 gm, dextrose, 5 gml'jsodium chlojon Saboura ds broth (4Q gim, dextrose and l'O'g'm. bacride and 2.5 gm. disodium phosphate) called B lll or to peptone)CaIIedQ SABT 'The cultures a r 'grown in tially neducing the po'ssibility of anallergic reaction and eliminating any interfefiejnce with the healingprocess h he ght sense b Wsh krh shallow layers of tnedia containedin flasks and held stationary until: X and extensive sporulation agents. P-riorto collectionof the growth mats, the ciiltures are killed by placing the-flasks info afcllosed oven under CO5 at-=l 2"/ C-.-'for three hours. Thefflaslt's are then cooled in the oven for an additional one hour and fifteen minutes, Culture broths are remo edby filtra- Itiojn jhgough Buchh'er funnels and the growth mats distilled Water, 7 The mats are then froin and 'lyophilizedand-the dry products gr'ou'nd in a more tar witha pestle under CO jNo attempt is made to pu- .rify :the chitin. Tw e l yerpairsof rats are 'used for "each test. some inflammation is observed on all treated wounds and infection on several. The increases in ,wound healing obtainedmay be all the more significant in View of these adverse" factors.

100 grams of dried fungus material (obtained from Penicillium fungus of Example 4, cultured on a BHI medium, sterilized by boiling the fungus with the medium and then filtering, washing with distilled water and drying the fungus material) is defatted by extracting the solvent-soluble fatty materials with 1000 ml. chloroform at room temperature. The chloroform is removed by filtering and then drying at reduced pressure in a vacuum desiccator.

The defatted fungus material is treated with 2000 ml.

1.0 N-NaOl-l solution for 18 hours at room temperature. The material is then acidified with HCl. Thereafter the material is dialyzed in distilled water until the wash water is free from chlorine ions. This procedure is repeated until a substantially purified material is obtained. The material is dried in vacuum below 50C and is a gray, friable mass.

The dried material is ground in a laboratory mortar and screened through a 400 mesh standard screen.

When the screened material is applied to 20 test rats of '20 pairs of rats there is obtained an average of about 25% increase in the wound healing of the treated rats over the untreated control rats.

EXAMPLE 7 Lobster shell chitin is purified by first slurrying it in -l% aqueous NaOH for minutes at 80C, then it is washed, drained and slurried in HCl for 5 minutes at 80C, drained, slurried in water, the pH of the water adjusted to 6 with dilute aqueous NaO l-l, and finally drained and dried.

The dried chitin material is pulverized to a fineness of about 40 microns. The material shows an average 25% increase in the wound healing over the untreated control rats.

Although the present invention has been decribed in conjunction with preferred embodiments, it is to be understood that modifications andavariations may be resorted to without departing from the spirit and scope thereof, as those skilled in the art will readily understand.

What is claimed is:

l. A process for facilitating healing of a wound in a mammal which comprises applying as a wound healing aid at the situs of the wound a wound-healing amount of a woven fabric structure including fibers selected from the group consisting of chitin and an N-acetylated partially depolymerized chitin.

2. A process according to claim 1 wherein said wound healing aid is in the form of a bandage including chitin fibers.

3. A process according to claim 1 wherein said fibers are used in the form of sutures.

4. A process according to claim 1, wherein said a wound healing aid is in the form of a dressing including chitin fibers.

5. A process according to claim 1, wherein said wound healing aid is in the form of a bandage including N-acetylated partially depolymerized chitin fibers 6. A process for facilitating healing of a wound in a mammal which comprises applying as a wound healing aid at the situs of the wound a wound-healing amount of a non-woven fabric structure including fibers selected from the group consisting of chitin ancl N- acetylated partially depolymerized chitin.

7. A process according to claim 6, wherein said wound healing aid is in the form of a bandage including N-acetylated partially depolymerized chitin fibers.

8. A process according to claim 6, wherein said wound healing aid is in the form of a dressing including 

1. A PROCESS FOR FACILITATING HEALING OF A WOUND IN A MAMMAL WHICH COMPRISES APPLYING AS A WOUND HEALING AID AT THE SITUS OF THE WOUND A WOUND-HEALING AMOUNT OF A WOVEN FABRIC STRUCTURE INCLUDING FIBERS SELECTED FROM THE GROUP CONSISTING OF CHITIN AND AN N-ACCTYLTED PARTIALLY DEPOLYMERIZED CHITIN.
 2. A process according to claim 1 wherein said wound healing aid is in the form of a bandage including chitin fibers.
 3. A process according to claim 1 wherein said fibers are used in the form of sutures.
 4. A process according to claim 1, wherein said wound healing aid is in the form of a dressing including chitin fibers.
 5. A process according to claim 1, wherein said wound healing aid is in the form of a bandage including N-acetylated partially depolymerized chitin fibers.
 6. A process for facilitating healing of a wound in a mammal which comprises applying as a wound healing aid at the situs of the wound a wound-healing amount of a non-woven fabric structure including fibers selected from the group consisting of chitin and N-acetylated partially depolymerized chitin.
 7. A process according to claim 6, wherein said wound healing aid is in the form of a bandage including N-acetylated partially depolymerized chitin fibers.
 8. A process according to claim 6, wherein said wound healing aid is in the form of a dressing including chitin fibers.
 9. A process according to claim 6, wherein said wound healing aid is in the form of a dressing including N-acetylated partially depolymerized chitin fibers. 